Image capturing apparatus and focusing method thereof

ABSTRACT

In the image capturing apparatus, the optical path difference producing member is disposed on the second optical path. Thereby, it is possible to suppress the amount of light when an optical image which is focused at the front of an optical image made incident into the first imaging device (front focus) and an optical image which is focused at the rear thereof (rear focus) are respectively imaged at the second imaging device and also to secure the amount of light on image pickup by the first imaging device. Further, in the image capturing apparatus, a position of the first imaging region and a position of the second imaging region on the imaging area are reversed with respect to the axis P in association with reversal of a scanning direction of the sample. Therefore, despite the scanning direction of the sample, it is possible to obtain a deviation direction of the focus position under the same conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/398,011, now U.S. Pat. No. 9,921,392, filed on Oct. 30, 2014, theentire contents of which is incorporated herein by reference. U.S.application Ser. No. 14/398,011 is a § 371 national stage application ofPCT/JP2013/050853, filed on Jan. 17, 2013, which claims priority toJapanese Patent Application No. 2011-277539. The entire contents of eachof the aforementioned applications are hereby incorporated by referencein their entirety.

TECHNICAL FIELD

The present invention relates to an image capturing apparatus which isused for capturing images of a sample, etc., and also relates to afocusing method thereof.

BACKGROUND ART

Image capturing apparatuses include a virtual microscope apparatus inwhich, for example, an imaging region of a sample is in advance dividedinto a plurality of regions to image the divided regions at a highmagnification and, thereafter, to synthesize the regions. In capturingimages by using the virtual microscope as described above,conventionally, as conditions for picking up images of a sample such asa biological sample, a focus map which covers an entire region of thesample is set to capture images of the sample, while focus control isperformed based on the focus map.

In preparation of the focus map, at first, an image capturing apparatusequipped with a macro optical system is used to capture an entire sampleas a macro image. Next, the thus captured macro image is used to set animage pickup range of the sample and also the range is divided into aplurality of regions to set a focus obtaining position for each of thedivided regions. After the focus obtaining position has been set, thesample is transferred to the image capturing apparatus equipped with amicro optical system to obtain a focus position at the thus set focusobtaining position, thereby preparing a focus map with reference to thefocus position.

However, in preparation of the above-described focus maps, there hasbeen a problem that processing needs time. Further, suppression ofintervals and the number of focuses to be obtained would reduce the timenecessary for the processing. In this case, however, there has been aproblem of reduction in focus accuracy. Therefore, development ofdynamic focus for capturing images of a sample at a high magnification,with a focus position being obtained, is now underway. The dynamic focusis a method in which a present direction of the focus position deviatingfrom the height of an objective lens is detected based on a differencein light intensity or a difference in contrast between an optical imagewhich is focused at the front of an optical image made incident into animaging device for capturing an image (front focus) and an optical imagewhich is focused at the rear thereof (rear focus), thereby allowing theobjective lens to move in a direction at which the deviation iscancelled to capture an image.

A microscope system disclosed, for example, in Patent Document 1, isprovided with a second imaging unit which images a region at the frontof a region imaged by a first imaging unit, an automatic focusingcontrol unit which adjusts a focusing position of an objective lens atan imaging position of the first imaging unit based on an image pickedup by the second imaging unit, and a timing control unit whichsynchronizes timing at which a divided region moves from an imagingposition of the second imaging unit to the imaging position of the firstimaging unit with timing at which an image forming position of thedivided region imaged by the second imaging unit is positioned at animaging area of the first imaging unit depending on a distance betweenthe divided regions and a speed at which a sample moves. Further, in amicroscope apparatus disclosed, for example, in Patent Document 2 orPatent Document 3, a glass member is used to make a difference inoptical path length inside a light guiding optical system for focuscontrol.

CITATION LIST Patent Literature

-   [Patent Document 1] Japanese Patent Application Laid-Open No.    2011-081211-   [Patent Document 2] Japanese Patent Publication No. WO2005/114287-   [Patent Document 3] Japanese Patent Publication No. WO2005/114293

SUMMARY OF INVENTION Technical Problem

In the microscope system described in Patent Document 1, a half mirrorand a mirror are used to form an optical path difference optical system,by which light different in optical path length is made incident intoeach of two imaging regions of the second imaging unit. In theconventional microscope system, for example, a line sensor is used toconstitute a first imaging unit and a second imaging unit. In the linesensor, it is important to secure an amount of light for capturing aclear image due to short exposure time. However, in the conventionalmicroscope system, light is divided by the optical path differenceoptical system. Thus, there is posed such a problem that it is difficultto secure an amount of light.

Technical Problem

Further, in the conventional microscope system, an optical surface of anoptical path dividing unit is inclined, by which a region imaged by thesecond imaging unit is adjusted so as to be on the front side of thesample in a scanning direction with respect to a region imaged by thefirst imaging unit, thereby capturing in advance a direction at whichthe focus position will deviate. However, the above-describedconfiguration has difficulty in adjusting the optical surface, whichposes a problem. In particular, where the sample changes in scanningdirection, it is necessary to adjust the optical surface before or afterthe change in scanning direction, which may result in complicatedadjustment work. Further, the half mirror and the mirror are used toconstitute the optical path difference optical system. Thus, there hasalso been a problem that light which has passed through the optical pathdifference optical system is less likely to gather on an imaging area ofthe second imaging unit.

The present invention has been made in order to solve theabove-described problems, an object of which is to provide an imagecapturing apparatus which is simple in configuration and capable ofobtaining a direction at which a focus position deviates under the sameconditions, despite a scanning direction of a sample, and also toprovide a focusing method thereof.

Solution to Problem

In order to solve the above-described problems, the image capturingapparatus of the present invention is characterized by having a stage onwhich a sample is placed, a stage control unit which scans the stage ata predetermined speed, a light source which radiates light to thesample, a light guiding optical system including a light dividing unitwhich divides an optical image of the sample into a first optical pathfor capturing an image and a second optical path for focus control, afirst imaging unit which captures a first image by a first optical imagedivided into the first optical path, a second imaging unit whichcaptures a second image by a second optical image divided into thesecond optical path, a focus control unit which analyzes the secondimage to control a focus position of the image pickup by the firstimaging unit based on the analysis result, a region control unit whichsets a first imaging region and a second imaging region for capturing apartial image of the second optical image on an imaging area of thesecond imaging unit, and an optical path difference producing memberwhich is disposed on the second optical path to give an optical pathdifference to the second optical image along an in-plane direction ofthe imaging area, in which the optical path difference producing memberis disposed in such a manner that an optical path difference of thesecond optical image is symmetrical with respect to an axis of theimaging area orthogonal to a direction at which the second optical imagemoves in association with scanning of the sample, and the region controlunit reverses a position of the first imaging region and a position ofthe second imaging region on the imaging area with respect to the axisin association with reversal of the scanning direction of the sample.

In the image capturing apparatus, the optical path difference producingmember is disposed on the second optical path. Thereby, at the firstimaging region and the second imaging region of the second imaging unit,it is possible to image respectively an optical image which is focusedat the front of an optical image made incident into the first imagingunit (front focus) and an optical image which is focused at the rearthereof (rear focus). In the image capturing apparatus, it is possibleto make a difference in optical path length without dividing light onthe second optical path for focus control. Therefore, an amount of lightat the second optical path necessary for obtaining information on afocus position can be suppressed to secure an amount of light on theimage pickup by the first imaging unit. Further, in the image capturingapparatus, it is possible to reverse the position of the first imagingregion and the position of the second imaging region on the imaging areawith respect to the axis of the imaging area in association withreversal of the scanning direction of the sample. Therefore, despite thescanning direction of the sample, it is possible to obtain a directionat which the focus position deviates under the same conditions.

It is also preferable that a region into which an optical image is madeincident which is conjugate to the first optical image made incidentinto the first imaging unit is substantially in alignment with the axisof the imaging area on the imaging area. Thereby, even where the sampleis reversed in scanning direction, it is possible to keep a positionalrelationship between the first imaging region, the second imaging regionand the region into which an optical image is made incident which isconjugate to the first optical image on the imaging area.

It is also preferable that the axis of the imaging area is substantiallyin alignment with a central axis of the imaging area orthogonal to adirection at which the second optical image moves in association withscanning of the sample. In this case, it is possible to reliably reversethe position of the first imaging region and the position of the secondimaging region on the imaging area in association with reversal of thescanning direction of the sample.

Still further, it is preferable that the optical path differenceproducing member is a flat plate member which is disposed so as tooverlap at least on a part of the imaging area and that the regioncontrol unit sets the first imaging region and the second imaging regionrespectively to give a region which will overlap on the flat platemember and a region which will not overlap on the flat plate member inorder to avoid a shadow of the second optical image by an edge part ofthe flat plate member. In this case, use of the flat plate memberenables the optical path difference producing member to be simple inconfiguration. Further, the edge part of the flat plate member forms theshadow of the second optical image at the imaging area of the secondimaging device. Therefore, the first imaging region and the secondimaging region are set so as to avoid the shadow, thus making itpossible to secure accurate control of the focus position.

It is also preferable that the optical path difference producing memberis a member having a part which undergoes a continuous change inthickness along an in-plane direction of the imaging area and that theregion control unit sets the first imaging region and the second imagingregion so as to overlap on the part of the optical path differenceproducing member which is different in thickness. In this case,adjustment of a position of the first imaging region and that of thesecond imaging region makes it possible to adjust freely an intervalbetween the front focus and the rear focus. Thereby, it is possible todetect a focus position of the sample at high accuracy.

It is also preferable that there are provided an objective lens whichfaces to a sample and an objective lens control unit which controls aposition of the objective lens relatively with respect to the samplebased on control by the focus control unit, in which the objective lenscontrol unit will not actuate the objective lens during analysis of thefocus position which is being performed by the focus control unit andwill allow the objective lens to move with respect to the sample in onedirection during analysis of the focus position which is not beingperformed by the focus control unit. In this case, since no change inpositional relationship will take place between the objective lens andthe sample during analysis of the focus position, it is possible tosecure analysis accuracy of the focus position.

It is also preferable that the region control unit sets waiting timefrom image pickup at the first imaging region to image pickup at thesecond imaging region based on a scanning speed of the stage and aninterval between the first imaging region and the second imaging region.Therefore, since light from the same position of the sample is madeincident into the first imaging region and the second imaging region, itis possible to control a focus position at high accuracy.

Further, the focusing method of the image capturing apparatus in thepresent invention is a focusing method of an image capturing apparatuswhich is characterized by having a light source which radiates light toa sample, a light conductive optical system including a light dividingunit which divides an optical image of the sample into a first opticalpath for capturing an image and a second optical path for focus control,a first imaging unit which captures a first image by the first opticalimage divided into the first optical path, a second imaging unit whichcaptures a second image by the second optical image divided into thesecond optical path, and a focus control unit which analyzes the secondimage to control a focus position of the image by the first imaging unitbased on an analysis result thereof, in which the first imaging regionand the second imaging region for capturing a partial image of thesecond optical image are set on the imaging area of the second imagingunit, an optical path difference producing member which gives an opticalpath difference to the second optical image along an in-plane directionof the imaging area is disposed on the second optical path in such amanner that the optical path difference of the second optical image issymmetrical with respect to the axis of the imaging area orthogonal to adirection at which the second optical image moves in association withscanning of the sample, and the region control unit reverses a positionof the first imaging region and a position of the second imaging regionon the imaging area with respect to the axis of the imaging area inassociation with reversal of the scanning direction of the sample.

In the focusing method of the image capturing apparatus, the opticalpath difference producing member is disposed on the second optical path.Thereby, at the first imaging region and the second imaging region ofthe second imaging unit, it is possible to image respectively an opticalimage which is focused at the front of an optical image made incidentinto the first imaging unit (front focus) and an optical image which isfocused at the rear thereof (rear focus). In the focusing method, it ispossible to make a difference in optical path length without dividinglight on the second optical path for focus control. Therefore, theamount of light at the second optical path necessary for obtaininginformation on a focus position can be suppressed to secure the amountof light on image pickup by the first imaging unit. Further, in thefocusing method, the position of the first imaging region and theposition of the second imaging region on the imaging area are reversedwith respect to the axis in association with reversal of the scanningdirection of the sample. Therefore, despite the scanning direction ofthe sample, it is possible to obtain a deviation direction of the focusposition under the same conditions.

It is also preferable that a region into which an optical image is madeincident which is conjugate to the first optical image made incidentinto the first imaging unit on the imaging area is made substantially inalignment with the axis of the imaging area. Thereby, even where thesample is reversed in scanning direction, it is possible to keep apositional relationship between the first imaging region, the secondimaging region and the region into which an optical image is madeincident which is conjugate to the first optical image on the imagingarea.

It is preferable that the axis of the imaging area is made substantiallyin alignment with the central axis of the imaging area orthogonal to adirection at which the second optical image moves in association withscanning of the sample. In this case, it is possible to reliably reversethe position of the first imaging region and the position of the secondimaging region on the imaging area in association with reversal of thescanning direction of the sample.

It is also preferable that as the optical path difference producingmember, there is used a flat plate member which is disposed so as tooverlap at least on a part of the imaging area and in order to avoid ashadow of the second optical image by an edge part of the flat platemember, the first imaging region and the second imaging region are setby the region control unit respectively so as to give a region whichwill overlap on the flat plate member and a region which will notoverlap on the flat plate member. In this case, use of the flat platemember enables the optical path difference producing member to be madesimple in configuration. Further, the edge part of the flat plate memberforms the shadow of the second optical image at an imaging area of thesecond imaging device. Therefore, the first imaging region and thesecond imaging region are set so as to avoid the shadow, thus making itpossible to secure accurate control of the focus position.

It is also preferable that as the optical path difference producingmember, there is used a member which has a part undergoing a continuouschange in thickness along an in-plane direction of the imaging area andthat the first imaging region and the second imaging region are set bythe region control unit so as to overlap on the part of the optical pathdifference producing member which is different in thickness. In thiscase, adjustment of a position of the first imaging region and that ofthe second imaging region makes it possible to freely adjust an intervalbetween the front focus and the rear focus. It is, thereby, possible todetect a focus position of the sample at high accuracy.

It is also preferable that the image capturing apparatus is providedwith an objective lens which faces to a sample and an objective lenscontrol unit which controls a position of the objective lens relativelywith respect to the sample based on control by the focus control unit,in which the objective lens control unit will not drive the objectivelens during analysis of the focus position which is being performed bythe focus control unit and will allow the objective lens to move withrespect to the sample in one direction during analysis of the focusposition which is not being performed by the focus control unit. Sinceno change in positional relationship will take place between theobjective lens and the sample during analysis of the focus position, itis possible to secure analysis accuracy of the focus position.

It is also preferable that waiting time from image pickup at the firstimaging region to image pickup at the second imaging region is set bythe region control unit based on a scanning speed of the stage and aninterval between the first imaging region and the second imaging region.Therefore, since light from the same position of the sample is madeincident into the first imaging region and the second imaging region, itis possible to control the focus position at high accuracy.

Advantageous Effects of Invention

The present invention is able to obtain a direction at which the focusposition deviates by a simple configuration according to the scanningdirection of the sample.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a drawing which shows one embodiment of a macro imagecapturing device which constitutes an image capturing apparatus of thepresent invention.

FIG. 2 is a drawing which shows one embodiment of a micro imagecapturing device which constitutes the image capturing apparatus of thepresent invention.

FIG. 3 is a block diagram which shows functional components of the imagecapturing apparatus.

FIG. 4 is a drawing which shows an analysis result of a contrast valuewhere a distance to the surface of a sample is in agreement with thefocal length of an objective lens.

FIG. 5 is a drawing which shows an analysis result of a contrast valuewhere a distance to the surface of the sample is longer than the focallength of the objective lens.

FIG. 6 is a drawing which shows an analysis result of a contrast valuewhere a distance to the surface of the sample is shorter than the focallength of the objective lens.

FIG. 7 is a drawing which shows one example of an optical pathdifference producing member and a second imaging device (forwarddirection).

FIG. 8 is a drawing which shows one example of the optical pathdifference producing member and the second imaging device (reversedirection).

FIG. 9 is a drawing which shows another example of the optical pathdifference producing member and the second imaging device (forwarddirection).

FIG. 10 is a drawing which shows another example of the optical pathdifference producing member and the second imaging device (reversedirection).

FIG. 11 is a drawing which shows a modified example of the optical pathdifference producing member.

FIG. 12 is a drawing which shows a relationship of the distance betweenthe objective lens and the surface of the sample with respect toscanning time of a stage.

FIG. 13 is a drawing which shows control of a scanning direction of thestage by a stage control portion.

FIG. 14 is a drawing which shows control of a scanning speed of thestage by the stage control portion.

FIG. 15 is a flow chart which shows motions of the image capturingapparatus.

DESCRIPTION OF EMBODIMENTS

Hereinafter, a description will be given in detail of preferredembodiments of the image capturing apparatus and the focusing method ofthe image capturing apparatus in the present invention with reference todrawings.

FIG. 1 is a drawing which shows one embodiment of the macro imagecapturing device which constitutes the image capturing apparatus of thepresent invention. FIG. 2 is a drawing which shows one embodiment of themicro image capturing device which constitutes the image capturingapparatus of the present invention. As shown in FIG. 1 and FIG. 2, animage capturing apparatus M is constituted with a macro image capturingdevice M1 for capturing a macro image of a sample S and a micro imagecapturing device M2 for capturing a micro image of the sample S. Theimage capturing apparatus M is an apparatus which sets, for example, aplurality of line-shaped divided regions 40 with respect to the macroimage captured by the macro image capturing device M1 (refer to FIG. 13)and produces a virtual micro image by capturing and synthesizing each ofthe divided regions 40 by the micro image capturing device M2 at a highmagnification.

As shown in FIG. 1, the macro image capturing device M1 is provided witha stage 1 which supports the sample S. The stage 1 is an XY stage whichis actuated in a horizontal direction by a motor or an actuator such asa stepping motor (pulse motor) or a piezo actuator, for example. Thesample S which is observed by using the image capturing apparatus M is,for example, a biological sample such as cells and placed on the stage 1in a state of being sealed on a slide glass. The stage 1 is actuatedinside the XY plane, by which an imaging position with respect to thesample S is allowed to move.

The stage 1 is able to move back and forth between the macro imagecapturing device M1 and the micro image capturing device M2 and providedwith functions to deliver the sample S between the devices. It isacceptable that when a macro image is captured, an entire image of thesample S is picked up at one time or the sample S is divided into aplurality of regions to pick up each of the images. It is alsoacceptable that the stage 1 is installed both on the macro imagecapturing device M1 and on the micro image capturing device M2.

A light source 2 which radiates light to the sample S and a condensinglens 3 which concentrates light from the light source 2 at the sample Sare disposed on a bottom of the stage 1. It is acceptable that the lightsource 2 is disposed so as to radiate light obliquely to the sample S.Further, a light guiding optical system 14 which guides an optical imagefrom the sample S and an imaging device 5 which images the optical imageof the sample S are disposed on an upper face of the stage 1. The lightguiding optical system 14 is provided with an image forming lens 6 whichforms the optical image from the sample S at an imaging area of theimaging device 5. Still further, the imaging device 5 is an area sensorwhich is capable of capturing, for example, a two-dimensional image. Theimaging device 5 captures an entire image of the optical image of thesample S made incident into the imaging area via the light guidingoptical system 14 and is housed at a virtual micro image housing portion39 to be described later.

As shown in FIG. 2, the micro image capturing device M2 is provided onthe bottom of the stage 1 with a light source 12 and a condensing lens13, as with the macro image capturing device M1. Further, a lightguiding optical system 14 which guides an optical image from the sampleS is disposed on the upper face of the stage 1. The optical system whichradiates light from the light source 12 to the samples may include anexcitation light radiating optical system which radiates excitationlight to the sample S and a dark-field illuminating optical system whichcaptures a dark-field image of the sample S.

The light guiding optical system 14 is provided with an objective lens15 disposed so as to face to the sample S and a beam splitter (lightdividing unit) 16 disposed at a rear stage of the objective lens 15. Theobjective lens 15 is provided with a motor and an actuator such as astepping motor (pulse motor) and a piezo actuator for actuating theobjective lens 15 in a Z direction orthogonal to a face on which thestage 1 is placed. A position of the objective lens 15 in the Zdirection is changed by these actuation units, thus making it possibleto adjust a focus position of image pickup when an image of the sample Sis captured. It is acceptable that the focus position is adjusted bychanging a position of the stage 1 in the Z direction or by changingpositions of both the objective lens 15 and the stage 1 in the Zdirection.

The beam splitter 16 is a portion which divides an optical image of thesample S into a first optical path L1 for capturing an image and asecond optical path L2 for focus control. The beam splitter 16 isdisposed at an angle of approximately 45 degrees with respect to anoptical axis from the light source 12. In FIG. 2, an optical pathpassing through the beam splitter 16 is given as the first optical pathL1, while an optical path reflected at the beam splitter 16 is given asthe second optical path.

On the first optical path L1, there are disposed an image forming lens17 which forms an optical image of the sample S (first optical image)which has passed through the beam splitter 16 and a first imaging device(first imaging unit) 18 in which an imaging area is disposed at an imageforming position of the image forming lens 17. The first imaging device18 is a device which is capable of capturing a one-dimensional image(first image) by the first optical image of the sample S, including, forexample, a two-dimensional CCD sensor which is capable of realizing, TDI(time delay integration) actuation. The first imaging device 18 has astructure formed by combining, for example, two TDI sensors different intransfer direction and in which light receiving portions, each of whichhas plural stages of light receiving arrays, are positioned at thecenter of the imaging area. It is noted that the first imaging device 18may be a line sensor.

Further, in a method which captures sequentially images of the sample S,with the stage 1 controlled at a constant speed, the first imagingdevice 18 may be a device such as a CMOS sensor or a CCD sensor which iscapable of capturing a two-dimensional image. First images picked up bythe first imaging device 18 are sequentially stored at a temporarystorage memory such as a lane buffer, thereafter, compressed and outputat an image producing portion 38 to be described later.

On the other hand, on the second optical path L2, there are disposed aview-field adjusting lens 19 which contracts an optical image of asample reflected by the beam splitter 16 (second optical image) and asecond imaging device (second imaging unit) 20. Further, at a frontstage of the second imaging device 20, there is disposed an optical pathdifference producing member 21 which gives an optical path difference tothe second optical image. It is preferable that the view-field adjustinglens 19 is constituted in such a manner that the second optical image isformed at the second imaging device 20 in a dimension similar to that ofthe first optical image.

The second imaging device 20 is a device which is capable of capturing atwo-dimensional image (second image) by the second optical image of thesample S, including, for example, sensors such as a CMOS (complementarymetal oxide semiconductor) and a CCD (charge coupled device). It is alsoacceptable that a line sensor is used.

The imaging area 20 a of the second imaging device 20 is disposed so asto be substantially in alignment with an XZ plane orthogonal to thesecond optical path L2. The second optical image is projected on theimaging area 20 a. The second optical image is such that where thesample S is scanned by the stage 1 in the forward direction, the imagingarea 20 a will move in a +Z direction and where the sample S is scannedin the reverse direction, the imaging area 20 a will move in a −Zdirection. The first imaging region 22A and the second imaging region22B, each of which captures a partial image of the second optical image,are set on the imaging area 20 a (for example, refer to FIG. 7). Thefirst imaging region 22A and the second imaging region 22B are set in adirection perpendicular to a direction (scanning direction: Z direction)at which the second optical image moves on the imaging area 20 a inassociation with scanning of the sample S. Further, the first imagingregion 22A and the second imaging region 22B are set, with apredetermined interval kept, both of which captures a part of the secondoptical image in a line shape. Thereby, an optical image which is at thesame region as that of the first optical image of the sample S capturedby the first imaging device 18 can be captured as the second opticalimage at the first imaging region 22A and the second imaging region 22B.

Thereby, the second imaging device 20 is able to capture an opticalimage which is focused at the front of a first optical image madeincident into the first imaging device 18 (front focus) and an opticalimage which is focused at the rear thereof (rear focus) based on theposition of the first imaging region 22A and that of the second imagingregion 22B. A focus difference between the front focus and the rearfocus is dependent on a difference between a thickness and an index ofrefraction of the optical path difference producing member 21 throughwhich the second optical image made incident into the first imagingregion 22A passes and a thickness and an index of refraction of theoptical path difference producing member 21 through which the secondoptical image made incident into the second imaging region 22B passes.

The optical path difference producing member 21 is a glass member whichgives an optical path difference to the second optical image along anin-plane direction of the imaging area 20 a. The optical path differenceproducing member 21 is disposed in such a manner that an optical pathdifference of the second optical image is symmetrical with respect tothe axis P of the imaging area 20 a orthogonal to a direction at whichthe second optical image moves in association with scanning of thesample S. Further, it is preferable that the optical path differenceproducing member 21 is disposed in such a manner that a face which facesto the second imaging device 20 is parallel with the imaging area (lightreceiving face) 20 a of the second imaging device. Thereby, it ispossible to reduce deflection of light by the face which faces to thesecond imaging device 20 and also to secure the amount of light which isreceived by the second imaging device 20. There will be described latera shape of the optical path difference producing member 21 and anexample in which the first imaging region 22A and the second imagingregion 22B are disposed on the imaging area 20 a.

FIG. 3 is a block diagram which shows functional components of the imagecapturing apparatus. As shown in the diagram, the image capturingapparatus M is provided with a computer system having housing portionssuch as a CPU, a memory, a communication interface and a hard disk, anoperation portion 31 such as a keyboard, a monitor 32 etc. Thefunctional components of the control portion 33 include a focus controlportion 34, a region control portion 35, an objective lens controlportion 36, a stage control portion 37, an image producing portion 38and a virtual micro image housing portion 39.

The focus control portion 34 is a portion which analyzes a second imagecaptured by the second imaging device 20 to control a focus position ofan image picked up by the first imaging device 18 based on the analysisresult. More specifically, the focus control portion 34 first determinesa difference between a contrast value of the image captured at the firstimaging region 22A and a contrast value captured at the second imagingregion 22B in the second imaging device 20.

Here, as shown in FIG. 4, where a focus position of the objective lens15 is in alignment with the surface of the sample S, an image contrastvalue of the front focus captured at the first imaging region 22A issubstantially in agreement with an image contrast value of the rearfocus captured at the second imaging region 22B. Thereby, a differencevalue between them is almost zero.

On the other hand, as shown in FIG. 5, where a distance to the surfaceof the sample S is longer than a focal length of the objective lens 15,an image contrast value of the rear focus captured at the second imagingregion 22B is greater than an image contrast value of the front focuscaptured at the first imaging region 22A. Therefore, a difference valuebetween them is a positive value. In this case, the focus controlportion 34 outputs instruction information to the objective lens controlportion 36 so as to be actuated in a direction at which the objectivelens 15 is brought closer to the sample S.

Further, as shown in FIG. 6, where a distance to the surface of thesamples is shorter than a focal length of the objective lens 15, animage contrast value of the rear focus captured at the second imagingregion 22B is smaller than an image contrast value of the front focuscaptured at the first imaging region 22A. Therefore, a difference valuebetween them is a negative value. In this case, the focus controlportion 34 outputs instruction information to the objective lens controlportion 36 so as to be actuated in a direction at which the objectivelens 15 is brought away from the sample S.

The region control portion 35 is a portion which controls a position ofthe first imaging region 22A and a position of the second imaging region22B at the imaging area 20 a of the second imaging device 20. The regioncontrol portion 35 sets at first the first imaging region 22A at apredetermined position based on operation from the operation portion 31and releases the setting of the first imaging region 22A after imagepickup at the first imaging region 22A. Then, the region control portion35 sets the second imaging region 22B, with a predetermined intervalkept in the Z direction from the first imaging region 22A, and releasesthe setting of the second imaging region 22B after image pickup at thesecond imaging region 22B.

At this time, waiting time W from image pickup at the first imagingregion 22A to image pickup at the second imaging region 22B is set basedon an interval d between the first imaging region 22A and the secondimaging region 22B and a scanning velocity v of the stage 1. Forexample, where the waiting time W is given as time W1 from the start ofimage pickup at the first imaging region 22A to the start of imagepickup at the second imaging region 22B, it is possible to determine thewaiting time with reference to a formula of W1=d/v−e1−st, withconsideration given to exposure time e1 of image pickup at the firstimaging region 22A and time st from release of the setting of the firstimaging region 22A to the setting of the second imaging region 22B.

Further, where waiting time W is given as waiting time W2 fromcompletion of image pickup at the first imaging region 22A to start ofimage pickup at the second imaging region 22B, it is possible todetermine the waiting time with reference to a formula of W2=d/v−st,with consideration given to time st from release of setting of the firstimaging region 22A to setting of the second imaging region 22B. Stillfurther, an interval d between the first imaging region 22A and thesecond imaging region 22B is set based on a difference in optical pathlength given by the optical path difference producing member 21.However, the interval d actually corresponds to a distance of the sampleS on a slide glass. Eventually, it is necessary to convert the intervald to the number of pixels of the second imaging region 22B. Where apixel size of the second imaging device 20 is expressed in terms ofAFpsz and magnification is expressed in terms of AFmag, the number ofpixels dpix corresponding to the interval d can be determined withreference to a formula of dpix=d÷(AFpsz/AFmag).

Further, the region control portion 35 reverses the position of thefirst imaging region 22A and the position of the second imaging region22B on the imaging area 20 a so as to give line symmetry to the axis Pof the imaging area 20 a in association of reversal of the scanningdirection of the sample S. This setting is carried out, whenevernecessary, depending on the shape and arrangement of the optical pathdifference producing member 21. For example, in the examples shown inFIG. 7 and FIG. 8, the optical path difference producing member 21Awhich is formed with a flat plate-shaped glass member is disposed so asto be in alignment with the axis P of the imaging area 20 a. A region22C into which an optical image is made incident which is conjugate tothe first optical image made incident into the first imaging device 18is positioned so as to be in alignment with the axis P of the imagingarea 20 a. In the present embodiment, the axis P of the imaging area 20a is kept in alignment with the central axis of the imaging area 20 aorthogonal to a direction at which the second optical image moves inassociation with scanning of the sample S.

Then, as shown in FIG. 7, where the sample S is scanned in the forwarddirection (+Z direction), at an upper half region of the imaging area20, the first imaging region 22A is set at a region which will notoverlap on the optical path difference producing member 21A, and thesecond imaging region 22B is set at a region which will overlap on theoptical path difference producing member 21A. Further, as shown in FIG.8, where the sample S is scanned in the reverse direction (−Zdirection), at a lower half region of the imaging area 20 a, the firstimaging region 22A is set at a region which will not overlap on theoptical path difference producing member 21A, and the second imagingregion 22B is set at a region which will overlap on the optical pathdifference producing member 21A.

There is a fear that an edge part E of the optical path differenceproducing member 21A may form a shadow 23 of the second optical image onthe imaging area 20 a. Therefore, it is preferable that the interval dbetween the first imaging region 22A and the second imaging region 22Bis made wider than the width of the shadow 23 and that the first imagingregion 22A and the second imaging region 22B are set at a position so asto avoid the shadow 23.

Further, in the examples shown in FIG. 9 and FIG. 10, an optical pathdifference producing member 21B composed of a prism-shaped glass memberwhich undergoes a continuous change in thickness toward the centerthereof in the Z direction is disposed so as to be in alignment with theaxis P of the imaging area 20 a. The optical path difference producingmember 21B changes in thickness so as to give a symmetric shape at thecenter of the axis P of the imaging area in which the thickness isswitched from an increase to a decrease along the Z direction. Theregion 22C into which an optical image is made incident which isconjugate to the first optical image made incident into the firstimaging device 18 is positioned so as to be in alignment with the centerof the imaging area 20 a in the Z direction. In the present embodimentas well, the axis P of the imaging area 20 a is kept in alignment withthe central axis of the imaging area 20 a orthogonal to a direction atwhich the second optical image moves in association with scanning of thesample S.

Moreover, as shown in FIG. 9, where the sample S is scanned in theforward direction (+Z direction), at an upper half region of the imagingarea 20 a, the first imaging region 22A and the second imaging region22B are set respectively so as to be on the upper side and on the lowerside. Further, as shown in FIG. 10, where the sample S is scanned in thereverse direction (−Z direction), at a lower half region of the imagingarea 20 a, the first imaging region 22A and the second imaging region22B are set respectively so as to be on the lower side and on the upperside.

Moreover, it is acceptable that, as the optical path differenceproducing member 21, for example, as shown in FIG. 11(a), there is usedthe optical path difference producing member 21C whose central part inthe Z direction is constant in thickness and which decreases inthickness moving towards the outside the both ends thereof in the Zdirection. For example, as shown in FIG. 11(b), it is also acceptablethat an optical path difference producing member 21D composed of twoglass members, each of which has a triangular prism-shaped crosssection, is disposed so as to decrease in thickness moving towards thecenter of the imaging area 20 a from both ends thereof in the Zdirection.

Where there is set a position of the region 22C into which an opticalimage is made incident which is conjugate to the first optical imagemade incident into the first imaging device 18 on the imaging area 20 a,a distance between the stage 1 and the objective lens 15 is at firstfixed to adjust a position of the stage 1 in such a manner that a crossline of a calibration slide is positioned at the center of a visualfield of the first imaging device 18. Then, the second imaging device 20is adjusted for its back focus in such a manner that the cross line ofthe calibration slide comes into a visual field of the second imagingdevice 20. Finally, an in-plane direction position of the second imagingdevice 20 is adjusted in such a manner that the cross line of thecalibration slide is positioned at a desired site of the imaging area 20a of the second imaging device 20.

The objective lens control portion 36 is a portion which controlsactuation of the objective lens 15. Upon receiving instructioninformation output from the focus control portion 34, the objective lenscontrol portion 36 actuates the objective lens 15 in the Z direction inaccordance with contents of the instruction information. It is, thereby,possible to adjust a focus position of the objective lens 15 withrespect to the sample S.

The objective lens control portion 36 will not actuate the objectivelens 15 during analysis of the focus position which is being performedby the focus control portion 34 and will actuate the objective lens 15only in one direction along the Z direction until start of analysis of anext focus position. FIG. 12 is a drawing which shows a relationship ofthe distance between the objective lens and the surface of the samplewith respect to scanning time of the stage. As shown in the drawing,during scanning of the sample S, there will take place alternately ananalysis period A of the focus position and an objective lens actuationperiod B based on an analysis result thereof. As described so far, nochange in positional relationship takes place between the objective lens15 and the sample S during analysis of the focus position, thus makingit possible to secure analysis accuracy of the focus position.

The stage control portion 37 is a portion which controls actuation ofthe stage 1. More specifically, the stage control portion 37 allows thestage 1 on which the sample S is placed to be scanned at a predeterminedspeed based on operation by the operation portion 31. The stage 1 isscanned, by which an imaging field of the sample S moves relatively andsequentially at the first imaging device 18 and the second imagingdevice 20. Scanning of the stage 1 is, as shown in FIG. 13, for example,bi-directional scanning in which after completion of scanning of onedivided region 40, the stage 1 is allowed to move in a directionorthogonal to the scanning direction and a next divided region 40 isscanned in the opposite direction.

Although the stage 1 is scanned at a constant speed while images arecaptured, actually, immediately after the start of scanning, there is aperiod during which the scanning speed is unstable due to influences ofvibrations of the stage 1 etc. Thus, as shown in FIG. 14, it ispreferable that there is set a scanning width longer than the dividedregion 40 and an acceleration period C for accelerating the stage 1, astabilization period D for stabilizing a scanning speed of the stage 1and a slowing-down period F for slowing down the stage 1 are allowed totake place individually when scanning is performed outside the dividedregion 40. It is, thereby, possible to capture an image insynchronization with a constant speed period E during which the stage 1is scanned at a constant speed. It is acceptable that image pickup isstarted during the stabilization period D and a data part obtainedduring the stabilization period D is deleted after the image has beencaptured. The above-described method is desirable when used for animaging device which requires void reading of data.

The image producing portion 38 is a portion at which a captured image issynthesized to produce a virtual micro image. The image producingportion 38 receives sequentially first images output from the firstimaging device 18, that is, images of individual divided regions 40,synthesizing these images to produce an entire image of the sample S.Then, based on the thus synthesized image, prepared is an image, theresolution of which is lower than that of the synthesized image, andhoused in a virtual micro image housing portion 39 by associating a highresolution image with a low resolution image. It is acceptable that animage captured by the macro image capturing device M1 is also associatedat the virtual micro image housing portion 39. It is also acceptablethat the virtual micro image is housed as one image or plurally dividedimages.

Next, a description will be given of motions of the above-describedimage capturing apparatus M.

FIG. 15 is a flow chart which shows motions of the image capturingapparatus M. As shown in the flow chart, at the image capturingapparatus M, at first, a macro image of the sample S is captured by themacro image capturing device M1 (Step S01). The thus captured macroimage is subjected to binarization by using, for example, apredetermined threshold value and, thereafter, displayed on a monitor32. A scope for capturing micro images from macro images is set byautomatic setting based on a predetermined program or manual setting byan operator (Step S02).

Then, the sample S is transferred to the micro image capturing device M2and focusing conditions are set (Step S03). Here, as described above,based on a scanning velocity v of the stage 1 and an interval d betweenthe first imaging region 22A and the second imaging region 22B, awaiting time W is set up to the start of image pickup at the secondimaging region 22B. It is more preferable that consideration is given toexposure time e1 of image pickup at the first imaging region 22A, timest from release of setting of the first imaging region 22A to setting ofthe second imaging region 22B etc.

After the focusing conditions have been set, scanning of the stage 1 isstarted to capture a micro image for each of the divided regions 40 ofthe sample S by the micro image capturing device M2 (Step S04). Incapturing the micro image by the first imaging device 18, at the secondimaging device 20, a deviating direction of the objective lens 15 withrespect to the sample S is analyzed based on a difference in contrastvalue between the front focus and the rear focus by the first imagingregion 22A and the second imaging region 22B, thereby adjusting aposition of the objective lens 15 in real time. After micro images havebeen captured completely for all the divided regions 40, the thuscaptured micro images are synthesized to produce a virtual micro image(Step S05).

As described so far, in the image capturing apparatus M, the opticalpath difference producing members 21 are disposed on the second opticalpath L2. Thereby, at the first imaging region 22A and the second imagingregion 22B of the second imaging device 20, it is possible to imagerespectively an optical image which is focused at the front of anoptical image made incident into the first imaging device 18 (frontfocus) and an optical image which is focused at the rear thereof (rearfocus). In the image capturing apparatus M, a difference in optical pathlength can be made without dividing light on the second optical path L2for focus control. Therefore, it is possible to suppress the amount oflight at the second optical path L2 necessary for obtaining informationon a focus position and to secure the amount of light on image pickup atthe first imaging device 18. Further, in the image capturing apparatusM, a position of the first imaging region 22A and a position of thesecond imaging region 22B on the imaging area 20 a are reversed so as togive line symmetry with respect to the axis P in association withreversal of the scanning direction of the sample S. Therefore, despitethe scanning direction of the sample S, it is possible to obtain adeviation direction of the focus position under the same conditions.

Further, in the image capturing apparatus M, based on a scanningvelocity v of the stage and an interval d between the first imagingregion 22A and the second imaging region 22B, a waiting time W is setfrom image pickup at the first imaging region 22A to image pickup at thesecond imaging region 22B. As a result, light from the same position ofthe sample S is made incident into the first imaging region 22A and thesecond imaging region 22B. Thus, it is possible to control a focusposition of the objective lens 15 at high accuracy.

Where, as the optical path difference producing member of the presentembodiment, there are used optical path difference producing members 21(21B to 21D) composed of a glass member which has a part changing inthickness along an in-plane direction of the imaging area 20 a at theimaging device 20, the region control portion 35 is used to adjust aposition of the first imaging region 22A and a position of the secondimaging region 22B. Thereby, it is possible to freely adjust an intervalbetween the front focus and the rear focus. Accordingly, for example,where a plural number of contrast peaks are found in an image picked upby the second imaging device 20 or where a peak is flat in shape, afocus difference between the front focus and the rear focus is adjusted,thus making it possible to detect a focus position of the sample S athigh accuracy.

Further, where, as the optical path difference producing member of thepresent embodiment, there are used optical path difference producingmembers 21 (21A) composed of a flat-plate like glass member, the opticalpath difference producing member 21 can be made simple in structure. Inthis case, an edge part E of the flat plate member forms a shadow 23 ofthe second optical image at the imaging area 20 a of the second imagingdevice 20. Therefore, the first imaging region 22A and the secondimaging region 22B are set so as to avoid the shadow 23, by which it ispossible to secure a focus position of the objective lens 15 at highaccuracy.

Further, in the above-described embodiment, the first imaging region 22Aand the second imaging region 22B are set so as to be positioned on thefront side of the sample S in the scanning direction on the imaging area20 a with respect to the region 22C into which an optical image is madeincident which is conjugate to the first optical image made incidentinto the first imaging device 18. Thereby, it is possible to obtain inadvance a deviation direction of the focus position in relation to animaging position of the first imaging device 18. The present inventionshall not be limited to the above-described mode but may include a modein which analysis is made for a deviation direction of the focusposition at an imaging position of the first imaging device 18.

In the above-described embodiment, there is exemplified an apparatus forproducing a virtual micro image. The image capturing apparatus of thepresent invention is, however, applicable to various types ofapparatuses, as long as the apparatuses are those in which an image iscaptured by scanning a sample at a predetermined speed by a stage etc.

REFERENCE SIGNS LIST

1 . . . stage, 12 . . . light source, 14 . . . light guiding opticalsystem, 15 . . . objective lens, 16 . . . beam splitter (light dividingunit), 18 . . . first imaging device (first imaging unit), 20 . . .second imaging device (second imaging unit), 20 a . . . imaging area, 21(21A to 21D) . . . optical path difference producing member, 22A . . .first imaging region, 22B . . . second imaging region, 34 . . . focuscontrol portion (focus control unit), 35 . . . region control portion(region control unit), 36 . . . objective lens control portion(objective lens control unit), E . . . edge part, L1 . . . first opticalpath, L2 . . . second optical path, M . . . image capturing apparatus,M1 . . . macro image capturing device, M2 . . . micro image capturingdevice, P . . . axis, S . . . sample.

The invention claimed is:
 1. An apparatus for capturing an image of asample, the apparatus comprising: a stage configured to support asample; an objective lens configured to face to the sample; a stagecontrol unit configured to move the stage at a moving speed; a beamsplitter optically coupled and configured to divide an optical image ofat least a portion of the sample into a first optical image and a secondoptical image; a first image sensor configured to capture the at least aportion of the first optical image; a second image sensor configured tocapture the at least a portion of the second optical image and provideimage data; a focus controller configured to analyze the image data soas to control a focus position of the objective lens based on theanalysis result; a region control unit of a computer system, configuredto set, at an imaging area of the second image sensor, a first imagingregion and a second imaging region so as to capture the at least theportion of the second optical image; and wherein the region control unitis configured to reverse a position of the first imaging region and aposition of the second imaging region on the imaging area with respectto an axis in association with reversal of the scanning direction of thesample, wherein the axis corresponds to the imaging area orthogonal to adirection at which the second optical image moves.
 2. The apparatus ofclaim 1, wherein on the imaging area, a region into which an opticalimage conjugate to the first optical image made incident into the firstimage sensor is in alignment with the axis of the imaging area.
 3. Theapparatus of claim 1, wherein the axis of the imaging area is disposedto be in a center of a Z direction of the second image sensor and asecond image area.
 4. The apparatus of claim 1, further comprising: anoptical path difference producing member configured to give an opticalpath difference to the second optical image along an in-plane directionof the imaging area.
 5. The apparatus of claim 1, further comprising: anobjective lens control unit configured to control a position of theobjective lens relatively with respect to the sample based on control bythe focus controller, wherein during an analysis of the focus positionby the focus controller, the objective lens control unit does notactuate the objective lens, and during an objective lens actuationperiod, the objective lens control unit moves the objective lens withrespect to the sample in one direction when analysis of the focusposition is not being performed.
 6. The apparatus of claim 1, whereinthe region control unit is configured to set waiting time from imagepickup at the first imaging region to image pickup at the second imagingregion based on a scanning speed of the stage and an interval betweenthe first imaging region and the second imaging region.
 7. A method forcapturing an image of a sample, the method comprising: by an objectivelens, acquiring an optical image of at least a portion of a samplesupported on a stage; dividing the optical image into a first opticalimage and a second optical image; capturing at least a portion of thefirst optical image; setting a first imaging region and a second imagingregion at an image area of an image sensor; by the first imaging regionand the second imaging region, capturing the at least the portion of thesecond optical image and providing image data; and analyzing the imagedata so as to control a focus position of the objective lens based onthe analysis result in an analysis period; and reversing a position ofthe first imaging region and a position of the second imaging region onthe imaging area with respect to an axis in association with reversal ofthe scanning direction, wherein the axis corresponds to the image areaorthogonal to a direction at which the second optical image moves. 8.The method of claim 7, wherein on the imaging area, a region into whichan optical image is made incident which is conjugate to the firstoptical image made incident into the image sensor is allowed to be inalignment with the axis of the imaging area.
 9. The method of claim 7,wherein the axis of the imaging area is allowed to be in alignment withthe central axis of the imaging area orthogonal to a direction at whichthe second optical image moves in association with scanning of thesample.
 10. The method of claim 7, further comprising: giving an opticalpath difference to the second optical image in such a manner that theoptical path difference of the second optical image is symmetrical withrespect to the axis of the imaging area orthogonal to a scanningdirection at which the second optical image moves in association withscanning of the sample.
 11. The method of claim 7, further comprising:controlling a position of the objective lens relatively with respect tothe sample based on control by the analysis result, wherein the controlis not actuated the objective lens in analysis period.
 12. The method ofclaim 7, further comprising: setting waiting time from image pickup atthe first imaging region to image pickup at the second imaging region.13. An apparatus for capturing an image of a sample, the apparatuscomprising: a stage configured to support a sample; an objective lensconfigured to face to the sample; a stage control unit configured tomove the stage at a moving speed; a beam splitter optically coupled andconfigured to divide an optical image of at least a portion of thesample into a first optical image and a second optical image; a firstimage sensor configured to capture the at least a portion of the firstoptical image; a second image sensor configured to capture the at leasta portion of the second optical image and provide image data; a focuscontroller configured to analyze the image data so as to control a focusposition of the objective lens based on the analysis result; a regioncontrol unit of a computer system, configured to set an imaging regionso as to capture the at least the portion of the second optical image;and wherein the region control unit is configured to reverse a positionof the imaging region with respect to the axis orthogonal to a directionat which the second optical image moves in association with reversal ofthe scanning direction of the sample.
 14. The apparatus of claim 13,wherein the axis corresponds to a region into which an optical imageconjugate to the first optical image made incident into the first imagesensor.
 15. The apparatus of claim 13, wherein the axis is disposed tobe in a center of image area of the second image sensor.
 16. Theapparatus of claim 13, further comprising: an optical path differenceproducing member configured to give an optical path difference to thesecond optical image along an in-plane direction of the imaging area.17. The apparatus of claim 13, further comprising: an objective lenscontrol unit configured to control a position of the objective lensrelatively with respect to the sample based on control by the focuscontroller, wherein during an analysis of the focus position by thefocus controller, the objective lens control unit does not actuate theobjective lens, and during an objective lens actuation period, theobjective lens control unit moves the objective lens with respect to thesample in one direction when analysis of the focus position is not beingperformed.
 18. A method for capturing an image of a sample, the methodcomprising: by an objective lens, acquiring an optical image of at leasta portion of a sample supported on a stage; dividing the optical imageinto a first optical image and a second optical image; capturing atleast a portion of the first optical image; setting an imaging region atan image area of an image sensor; by the imaging region, capturing theat least the portion of the second optical image and providing imagedata; and analyzing the image data so as to control a focus position ofthe objective lens based on the analysis result in an analysis period;and reversing a position of the imaging region with respect to an axisorthogonal to a direction at which the second optical image moves inassociation with reversal of the scanning direction.
 19. The method ofclaim 18, wherein the axis corresponds to a region into which an opticalimage conjugate to the first optical image made incident into the firstimage sensor.
 20. The method of claim 18, wherein the axis is disposedto be in a center of image area of the second image sensor.
 21. Themethod of claim 18, further comprising: determining an optical pathdifference to the second optical image along an in-plane direction ofthe imaging area.
 22. The method of claim 18, further comprising:controlling a position of the objective lens relatively with respect tothe sample based on control by a focus controller, wherein during ananalysis of the focus position by the focus controller, the objectivelens is stationary, and during an objective lens actuation period, theobjective lens moves with respect to the sample in one direction whenanalysis of the focus position is not being performed.